Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth's supporting structures. Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem. One bacterium that has attracted considerable interest is P. gingivalis as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas P. gingivalis is rarely recovered, and then in low numbers, from healthy sites. A proportional increase in the level of P. gingivalis in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease. The progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of P. gingivalis. These findings in both animals and humans suggest a major role for P. gingivalis in the development of adult periodontitis.
P. gingivalis is a black-pigmented, anaerobic, asaccharolytic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids. The microorganism has an absolute growth requirement for iron, preferentially in the form of haeme or its Fe(III) oxidation product haemin and when grown under conditions of excess haemin is highly virulent in experimental animals. A number of virulence factors have been implicated in the pathogenicity of P. gingivalis including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes. In particular, proteases have received a great deal of attention for their ability to degrade a broad range of host proteins including structural proteins and others involved in defence. The proteins that have been shown to be substrates for P. gingivalis proteolytic activity include collagen types I and IV, fibronectin, fibrinogen, laminin, complement and plasma clotting cascade proteins, α1-antitrypsin, α2-macroglobulin, antichymotrypsin, antithrombin III, antiplasmin, cystatin C, IgG and IgA. The major proteolytic activities associated with this organism have been defined by substrate specificity and are “trypsin-like”, that is cleavage on the carboxyl side of arginyl and lysyl residues and collagenolytic although other minor activities have been reported.
P. gingivalis trypsin-like proteolytic activity has been shown to degrade complement, generating biologically active C5a, impair the phagocytic and other functions of neutrophils by modifying surface receptors, and abrogate the clotting potential of fibrinogen prolonging plasma clotting time. The trypsin-like proteolytic activity of P. gingivalis also generates Fc fragments from human IgG1 stimulating the release of pro-inflammatory cytokines from mononuclear cells and is associated with vascular disruption and enhanced vascular permeation through the activation of the kallikrein-kinin cascade. P. gingivalis spontaneous mutants with reduced trypsin-like activity as well as wild-type cells treated with the trypsin-like protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone are avirulent in animal models. Further, it has been shown that P. gingivalis grown under controlled, haemin-excess conditions expressed more trypsin-like and less collagenolytic activity and were more virulent in mice relative to cells grown under haemin-limited but otherwise identical conditions.
There has been considerable endeavour to purify and characterise the trypsin-like proteases of P. gingivalis from cell-free culture fluids. Chen et al, (1992) [J Biol Chem 267:18896-18901] have purified and characterised a 50 kDa arginine-specific, thiol protease from the culture fluid of P. gingivalis H66 designated Arg-gingipain. A similar arginine-specific thiol protease has been disclosed in JP 07135973 and the amino acid sequence disclosed in WO 9507286 and in Kirszbaum et al, 1995 [Biochem Biophys Res Comm 207:424-431]. Pike et al (1994) [J Biol Chem 269:406-411] have characterised a 60 kDa lysine-specific cysteine proteinase from the culture fluid of P. gingivalis H66 designated Lys-gingipain and the partial gene sequence for this enzyme was disclosed in WO 9511298 and fully disclosed in WO 9617936. In addition, a cell surface protein complex of P. gingivalis comprising a 300 kDa complex of arginine-specific and lysine-specific proteases both containing adhesin domains is disclosed in U.S. Pat. No. 6,511,666.